The molecular mechanism by which polarity is established and maintained during Candida albicans hyphal tip growth remains largely unknown and this gap in knowledge has limited the development of models for how hyphae invade tissues how this can be prevented. The long-term goal associated with this research program is to understand how polarity is established at hyphal tips (hyphal guidance) and how hyphal guidance proteins mediate C. albicans contact sensing (thigmotropic) and invasive behavior. These objectives will be achieved in this proposal by pursuing the following three Specific Aims: 1) Identify proteins that are important for polarity establishment at hyphal tips;2) Determine the extent to which tip-localized polarity establishment proteins are involved in thigmotropism and invasion;and 3) Establish the mechanism by which the known hyphal guidance GTPase, Rsrlp, controls hyphal guidance. Under the first aim, a forward genetic screen will be used to identify cortically-localized polarity establishment proteins, (by localization of GFP fusions) and by analysis of polarity establishment phenotypes in strains lacking these tip-localized candidate proteins. Under the second aim, the ability of hyphal guidance proteins to mediate hyphal re-orientation in response to a ridge and epithelial cell damage will be determined. Under the third aim, genetic and biochemical interactions between Rsrlp and unique polarity proteins at the hyphal tip will be determined by a) localization of tip polarity structures (polarisome and Spitzenkorper) in strains lacking Rsrlp activity, b) co-immunoprecipitation analysis of physical interactions among Rsrlp and tip polarity proteins, and c) epistasis analysis of thigmotropic and invasive ability of strains lacking both Rsrlp and a second polarity establishment protein. The proposed research is significant because it will provide the knowledge needed to understand how hyphae grow in a directional manner and how this is related to a clinically relevant process - host tissue invasion. At the same time, the fundamental new knowledge obtained about polarity establishment in hyphae is expected to advance the general field of polarity responses in other filamentous fungi and in other polarized eukaryotic cell systems. This is an important and under-investigated area of C. albicans biology that has applicability to understanding the pathogenesis of tissue invasion during systemic C. albicans infections in immunocompromised patients.